The human papillomavirus (HPV) E6 oncoprotein is expressed in over 95 percent of cervical cancers. HPV E6, in complex with the cellular ubiquitin-protein ligase E6AP, targets the p53 tumor suppressor for degradation by the ubiquitin-mediated proteolytic system. Several lines of evidence indicate that HPV E6 has additional, p53-independent functions. Scribble and utrophin are two recently identified E6-dependent substrates of E6AP that are involved in cell polarity and membrane organization. The main goal of this proposal is to study the structure-function relationship of E6 proteins with respect to their role in facilitating E6AP-dependent ubiquitination. Structure-function analyses of ES proteins have resulted in conflicting and confusing results, and evidence strongly suggests that this may be due to problems expressing properly folded E6 proteins. HPV E6 proteins will be expressed in an optimized system, and a set of chimeric and mutated proteins will be screened for those that maintain characteristics of properly folded proteins. These proteins will be used to map determinants necessary for E6AP binding and ubiquitination of known E6/E6AP-dependent substrates. Characterized mutant HPV E6 proteins will be used in immortalization assays of primary keratinocytes to determine which functions are necessary for the ability of E6 to immortalize cells. These studies are significant because they will provide insights into the mechanism of E6-dependent ubiquitination of substrates and a greater understanding of the role of high-risk HPV E6 proteins in human cervical cancer.